Sulfonamide-metal complexes endowed with potent anti-Trypanosoma cruzi activity.

In this article, we describe that mononuclear complexes composed of (5-chloro-2-hydroxybenzylidene)aminobenzenesulfonamides (L1-3) of general formula (L2(M)2H2O, where M is Co, Cu, Zn, Ni or Mn) reduced epimastigote proliferation and were found cidal for trypomastigotes of Trypanosoma cruzi Y strain. Complexes C5 and C11 have IC50 of 2.7 ± 0.27 and 4.8 ± 0.47 µM, respectively, for trypomastigotes, when the positive control Nifurtimox, which is also an approved drug for Chagas disease, showed IC50 of 2.7 ± 0.25 µM. We tested whether these complexes inhibit the enzyme T. cruzi trypanothione reductase or acting as DNA binders. While none of these complexes inhibited trypanothione reductase, we observed some degree of DNA binding, albeit less pronounced than observed for cisplatin in this assay. Unfortunately, most of these complexes were also toxic for mouse splenocytes. Along with the present studies, we discuss a number of interesting structure-activity relationships and chemical features for these metal complexes, including computational calculations.

Cytokines and NO in American tegumentary leishmaniasis patients: profiles in active disease, after therapy and in self-healed individuals.

Studies suggest the influence of immune response on the successful treatment of American tegumentary leishmaniasis (ATL), and indicate the existence of protective immunity in self-healed patients. Thus, the aim of this work was to quantify interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL-) 10, IL-17, IL-22 and nitric oxide (NO) in culture supernatants of PBMC from patients with active disease (AD), after treatment (AT), and from self-healed (SH) and healthy subjects (CT), in response to Leishmania (Viannia) braziliensis insoluble antigen (AgIns). All groups of patients produced IFN-γ, indicating a predominant proinflammatory profile. AD and AT patients presented TNF-α levels, with a slight increase after therapy, whereas it was weakly quantified in SH. Interestingly, NO secretion was significant in these individuals, whereas IL-17 appeared in low levels and seems to be regulated by NO. Although IL-22 was detected in AD, its role is still questionable. The presence of IL-10 in all groups of patients suggests that the cytokine plays distinct roles in the disease. These results indicate that specific cellular immunity takes part against Leishmania, but with some similarities between the different clinical states herein described; these mediators seem to be necessary for the cure to occur.

Comparison of flow cytometry and indirect immunofluorescence assay in the diagnosis and cure criterion after therapy of American tegumentary leishmaniasis by anti-live Leishmania (Viannia) braziliensis immunoglobulin G.

The aim of this study was to compare the techniques of indirect immunofluorescence assay (IFA) and flow cytometry to clinical and laboratorial evaluation of patients before and after clinical cure and to evaluate the applicability of flow cytometry in post-therapeutic monitoring of patients with American tegumentary leishmaniasis (ATL). Sera from 14 patients before treatment (BT), 13 patients 1 year after treatment (AT), 10 patients 2 and 5 years AT were evaluated. The results from flow cytometry were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). The 1:256 sample dilution allowed us to differentiate individuals BT and AT. Comparative analysis of IFA and flow cytometry by ROC (receiver operating characteristic curve) showed, respectively, AUC (area under curve)=0.8 (95% CI=0.64-0.89) and AUC=0.90 (95% CI=0.75-0.95), demonstrating that the flow cytometry had equivalent accuracy. Our data demonstrated that 20% was the best cut-off point identified by the ROC curve for the flow cytometry assay. This test showed a sensitivity of 86% and specificity of 77% while the IFA had a sensitivity of 78% and specificity of 85%. The after-treatment screening, through comparative analysis of the technique performance indexes, 1, 2 and 5 years AT, showed an equal performance of the flow cytometry compared with the IFA. However, flow cytometry shows to be a better diagnostic alternative when applied to the study of ATL in the cure criterion. The information obtained in this work opens perspectives to monitor cure after treatment of ATL.

Cellular immune response evaluation of cutaneous leishmaniasis patients cells stimulated with Leishmania (Viannia) braziliensis antigenic fractions before and after clinical cure.

American cutaneous leishmaniasis (ACL) is a disease where susceptibility or resistance is dependent on T cell response. This is characterized by an increased in CD4⁺ T cells, capable of inducing opposite disease profiles, and CD8⁺ T cells, that are related to immuno protection. We characterized T lymphocytes from patients before and after treatment, patients that spontaneously healed and controls, also evaluating their production of IL-10, IL-4, TNF-α and IFN-γ, after stimulation with soluble/insoluble antigenic fractions of Leishmania (Viannia) braziliensis. We observed the production of suppressive cytokines in the early phase of leishmaniasis with significant presence CD4⁺ T cells, suggesting their connection with disease progression. After healing, the immune pattern observed was a type 1 response, what seems to be associated with cure and/or protection in the ACL. The results also showed that both fractions induced a specific immune response, contributing to the search for relevant antigens in this disease.

Evaluation of anti-lived and anti-fixed Leishmania (Viannia) braziliensis promastigote IgG antibodies detected by flow cytometry for diagnosis and post-therapeutic cure assessment in localized cutaneous leishmaniasis.

This study aims to investigate a flow cytometry performance-based methodology to detect anti-live (FC-ALPA-IgG) and anti-fixed (FC-AFPA-IgG) Leishmania (Viannia) braziliensis promastigote IgG as a means to monitor post-therapeutic cure of patients with localized cutaneous leishmaniasis (LCL). Serum samples from 30 LCL patients infected with L. (V.) braziliensis were assayed, comparing the IgG reactivity before and after specific treatment with pentavalent antimonial. Reactivities were reported as the percentage of positive fluorescent parasites (PPFP), using a PPFP of 60% as a cut-off value. In the serum dilution of 1:1024, the positive percentage of LCL serum sample for FC-ALPA-IgG and FC-AFPA-IgG was 86% and 90%, respectively, before treatment. Analysis of ∆PPFP that represents the difference between PPFP after and before treatment appeared as a new approach to monitor post-therapeutic IgG reactivity in LCL. Our data support the perspective of using FC-ALPA and FC-AFPA as a useful serologic tool for diagnosis and for post-therapeutic follow-up of LCL patients.

Immunomodulatory response of Cramoll 1,4 lectin on experimental lymphocytes.

Cramoll 1,4 is a lectin extracted from Cratylia mollis Mart. seeds that has shown antitumor and lymphocyte mitogenic activities in other studies. The aim of this work was to investigate, in vitro, the immunomodulatory activity of Cramoll 1,4 on experimental cultures of mice lymphocytes through cytotoxic assays, nitric oxide (NO) concentrations and IL-10 and IFN-γ production. Cramoll 1,4 did not show cytotoxic activity at 1-25 µg/mL concentrations, similar results were observed with concanavalin A (Con A) and phytohemagglutinin (PHA) lectins. The minimum production of IL-10 was observed in splenocytes cultivated with Con A, PHA and Cramoll 1,4 lectins. However, splenocytes treated with Cramoll 1,4 showed higher IFN-γ production in comparison with PHA and Con A (p < 0.05 for both). Production of NO was effectively suppressed in murine cells stimulated with the lectins and was only detected after 72 h for PHA in relation to non-stimulated lymphocytes (p < 0.05). Cramoll 1,4 was not toxic to murine lymphocytes, induced Th1 response through IFN-γ production and showed antiinflammatory activity through NO suppression. Therefore, Cramoll 1,4 can be considered a lectin with immunomodulatory activity.